Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia and remains incurable. The introduction of the CD20 antibody rituximab combined with chemotherapy (chemoimmunotherapy) was the first intervention that has been shown to impact survival of CLL patients. Building upon the success of antibody directed therapy in CLL, we have actively pursued development of similar therapies directed at alternative antigens expressed selectively on CLL cells. CD37 is a tetraspanin protein of uncertain function that is expressed predominately on mature B-cells. Our research group has worked with Trubion Pharmaceutics, Inc/Emergent Biosolutions to develop CD37 directed TRU-016, a small modular immune pharmaceutical similar to an antibody but lacking the CH1 domain. Prior pre-clinical work demonstrated TRU-016 to have potent direct and immune based killing of CLL cells and also in vivo activity in several lymphoma pre-clinical models. A first in man phase 1 clinical trial initiated with TRU-016 in previously treated CLL has demonstrated single agent activity and combination studies have now been initiated. During this early phase 1 development of TRU-016 we have continued pre-clinical work to discern its mechanism of action. Preliminary data described in this proposal demonstrate that TRU-016 ligation of the CD37 antigen causes recruitment and phosphorylation of SHP1 via a potential ITIM domain within the intracytplasmic domain. SHP1 activation leads to decreased PI3-Kinase activity and AKT phosphorylation concomitant with FOXO3A accumulation in the nucleus and induction of the pro-apoptotic gene BIM and CLL cell death. siRNA targeting either SHP1 or BIM antagonizes death induced by TRU-016. Interestingly, the C-terminal of the CD37 protein contains ITAM like motif with associated activation function. Deletion of this motif enhances TRU-016 induced cytotoxicity suggesting a potential negative regulatory role for the C-terminal in TRU-016 cytotoxic effect. Consistent with this, inhibition of pro-survival signaling by PI3K and BTK overcame the inhibitory effects of the C-terminal domain resulting in enhanced TRU-016 induced apoptosis. Moving forward, this proposal seeks to further characterize in detail the ITIM and ITAM function of CD37 activated by TRU-016, study the mechanisms of cross talk between the activation and inhibitory signaling relevant to TRU-016 response and pursue development of afucosylated TRU-016 (TRU-016GV) leading to rational combination strategies that will facilitate enhanced TRU-016 and TRU-016GV response. Knowledge gained from this proposal has potential to impact not only CLL but also lymphoma and acute lymphoblastic leukemia patients who may also derive benefit from TRU-016 or TRU-016GV. Additionally, mechanistic study of CD37 ITIM and ITAM dual signaling has relevance to general B-cell immunology. The senior investigative team formed to perform this project includes expertise to accomplish the proposed goals and support from a large internationally known leukemia program. Collectively, TRU-016 and its application to CLL therapy have potential to transform treatment of this disease. PUBLIC HEALTH RELEVANCE: The overarching goal of this proposal is to characterize CD37 mediated cell death and survival mechanisms, and to develop combination strategies to best enhance mechanism based CD37 targeted therapeutic efficacy of TRU-016, a novel small modular immunopharmaceutics in CLL and other CD37-expressing B-cell malignancies. Successful completion of this work would contribute to more effective use of this novel agent and lead to improved survival for patients with CLL and NHL. Successful completion of this work would contribute to more effective use of this novel agent and lead to improved survival for patients with CLL, NHL, and ALL.